人表皮生长因子受体酪氨酸激酶的克隆表达和酶抑制剂筛选模型的构建
摘要
表皮生长因子受体(epidermal growth factor receptor,EGFR)属受体酪氨酸激酶(receptor tyrosine kinase,RTK)家族成员。EGFR在与配基EGF结合后,诱导受体二聚化,并激活胞内区的酪氨酸激酶和发生自磷酸化反应。
人的EGFR基因定位于第7号染色体的短臂(7p12.3-p12.1),它的编码产物EGFR由1210个氨基酸组成,是一种分子量约为170 kDa跨膜糖蛋白。其中,712-979位属于酪氨酸激酶区。EGFR由含621个氨基酸残基构成的胞外域、23个氨基酸残基组成的跨膜区、和具有酪氨酸激酶活性由542个氨基酸残基形成的胞内结构域组成。
EGFR在细胞信号转导通路中具有十分重要的作用。许多肿瘤的发生、发展都与EGFR胞内酪氨酸激酶的异常表达密切相关。因此,EGFR胞内酪氨酸激酶的抑制剂有可能成为治疗肿瘤的有效药物。
本研究从人脐静脉内皮细胞(HUVEC)提取总RNA,采用RT-PCR获得EGFR酪氨酸激酶(EGFR-TK)催化域的编码基因。以pET-30a为载体,在E.coli BL21(DE3)中成功克隆并表达了EGFR-TK。采用Ni-NTA亲和层析对表达产物进行了纯化,通过对酶活性的测定,证明重组EGFR酪氨酸激酶蛋白具有利用ATP催化底物发生磷酸化反应的激酶活性。以该重组激酶为靶位构建了酶抑制剂筛选模型,对微生物代谢产物进行了筛选,初筛获得9株阳性菌株,为研究新型结构的抗肿瘤微生物药物奠定了基础。
The Epidermal growth factor receptor(EGFR) is a member of the receptor tyrosine kinase family(PTK).After binding with the ligand(EGF),the process of receptor dimerization,active tyrosine kinase and autophosphorylation has been induced in the intracellular domain.
The genetic locus of human EGFR encoding the protein consisted of 1210 amino acid residues,located on the chromosome 7p12.3-p12.1.As a transmembrane glycoprotein with Mw 170 kDa,the tyrosine kinase domain situated on between 712 and 979 amino acid.The molecular structure of EGFR consists of an amino-terminal extracellular domain with 621 amino acid residues,a single transmembrane-anchoring region containing 23 amino acid residues,and a carboxylterminal intracellular domain with tyrosine kinase(TK) activity consisting of 542 amino acid residues.
EGFR plays an essential role in the regulation of signal transduction in the cells.Due to the abnormal expression of EGFR-TK,the certain type cancers may developed and progressed.Based on that,the inhibitors of EGFR-TK could be effective medicines for the treatment of cancer.
In this study,the gene encoding the catalyzing domain of EGFR-TK was amplified by RT-PCR with the RNA of HUVCEs cells as the template,which was cloned and expressed in E.coli BL21(DE3) using pET30a as vector.The recombinant protein EGFR-TK was purified with an affinity chromatography(Ni-NTA),which was identified to be the tyrosine kinase catalyzing the substrate phosphorylated with ATP in the enzymatic reaction.With the EGFR-TK as target,the screening model of enzymatic inhibitors was constructed,and some positive strains has been selected from more than 400 microbe strains.It will lay the foundation studying novel medicine for the treatment of cancer originated from microbe.
人的EGFR基因定位于第7号染色体的短臂(7p12.3-p12.1),它的编码产物EGFR由1210个氨基酸组成,是一种分子量约为170 kDa跨膜糖蛋白。其中,712-979位属于酪氨酸激酶区。EGFR由含621个氨基酸残基构成的胞外域、23个氨基酸残基组成的跨膜区、和具有酪氨酸激酶活性由542个氨基酸残基形成的胞内结构域组成。
EGFR在细胞信号转导通路中具有十分重要的作用。许多肿瘤的发生、发展都与EGFR胞内酪氨酸激酶的异常表达密切相关。因此,EGFR胞内酪氨酸激酶的抑制剂有可能成为治疗肿瘤的有效药物。
本研究从人脐静脉内皮细胞(HUVEC)提取总RNA,采用RT-PCR获得EGFR酪氨酸激酶(EGFR-TK)催化域的编码基因。以pET-30a为载体,在E.coli BL21(DE3)中成功克隆并表达了EGFR-TK。采用Ni-NTA亲和层析对表达产物进行了纯化,通过对酶活性的测定,证明重组EGFR酪氨酸激酶蛋白具有利用ATP催化底物发生磷酸化反应的激酶活性。以该重组激酶为靶位构建了酶抑制剂筛选模型,对微生物代谢产物进行了筛选,初筛获得9株阳性菌株,为研究新型结构的抗肿瘤微生物药物奠定了基础。
The Epidermal growth factor receptor(EGFR) is a member of the receptor tyrosine kinase family(PTK).After binding with the ligand(EGF),the process of receptor dimerization,active tyrosine kinase and autophosphorylation has been induced in the intracellular domain.
The genetic locus of human EGFR encoding the protein consisted of 1210 amino acid residues,located on the chromosome 7p12.3-p12.1.As a transmembrane glycoprotein with Mw 170 kDa,the tyrosine kinase domain situated on between 712 and 979 amino acid.The molecular structure of EGFR consists of an amino-terminal extracellular domain with 621 amino acid residues,a single transmembrane-anchoring region containing 23 amino acid residues,and a carboxylterminal intracellular domain with tyrosine kinase(TK) activity consisting of 542 amino acid residues.
EGFR plays an essential role in the regulation of signal transduction in the cells.Due to the abnormal expression of EGFR-TK,the certain type cancers may developed and progressed.Based on that,the inhibitors of EGFR-TK could be effective medicines for the treatment of cancer.
In this study,the gene encoding the catalyzing domain of EGFR-TK was amplified by RT-PCR with the RNA of HUVCEs cells as the template,which was cloned and expressed in E.coli BL21(DE3) using pET30a as vector.The recombinant protein EGFR-TK was purified with an affinity chromatography(Ni-NTA),which was identified to be the tyrosine kinase catalyzing the substrate phosphorylated with ATP in the enzymatic reaction.With the EGFR-TK as target,the screening model of enzymatic inhibitors was constructed,and some positive strains has been selected from more than 400 microbe strains.It will lay the foundation studying novel medicine for the treatment of cancer originated from microbe.
引文
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10.钱军,秦叔逵。针对表皮生长因子受体的靶向治疗研究进展。中国药科大学学报,2004,35(3):285-288.
11.张勇,王渭池,李元。人可溶性白介素-4受体(sIL-4R)在大肠杆菌中的表达和纯化。中国生物化学与分子生物学报,2004,20(6):778-784.
12.Edit varkondi,Eszter Schafer,Gyorgyi bokonyi,et al.Comparison ofElisa-Based tyrosine kinase assays for screening EGFR inhibitors.Journal of receptors and signal transducrion,2005;25:45-56
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11.张勇,王渭池,李元。人可溶性白介素-4受体(sIL-4R)在大肠杆菌中的表达和纯化。中国生物化学与分子生物学报,2004,20(6):778-784.
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1.曹川,戴霞,李世荣。受体酪氨酸蛋白激酶的研究,医学综述,2008:14(12)
2. Shawver LK, Slamon D, Ullrich A. Smart drugs; tyrosine kinase inhibitors in cancer therapy. Cancer cell 2002;1(2):117-23
3. Joseph Schlessinger. Cell Signaling by Receptor Tyrosine Kinases. Cell, 2000;103:211 -225.
4. Yosef Yarden,Mark X,Sliwkowski. Untangling the ErbB Signalling Network. Molecular Cell Biology,2001;2:127-137
5. Wieduwilt, M. J. and M. M. Moasser. The epidermal growth factor receptor family: biology driving targeted therapeutics. Cell Mol.Life Sci. 2008;65 (10):1566-84
6. D. Graus-Porta, R.R. Beerli, J.M. Daly, N.E. Hynes. ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling. EMBO J. 1997;16 :1647—1655.
7. Sanderson, M.P., Dempsey, P.J. and Dunbar, A.J. Control of ErbB signaling through metallo protease mediated ectodomain shedding of EGF-like factor. Growth Factors. 2006;24:121-136.
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